Project summary/Abstract Despite the central role of androgen receptor (AR) in prostate cancer (CaP) progression, insights into the specific molecular mechanisms by which AR controls the cellular processes that drive CaP progression remain largely elusive. Lack of this knowledge is an important problem, because without it, development of novel therapeutic approaches that target specifically AR-dependent events underlying the lethal phenotype is unlikely. The long- term goal is to understand how the mechanism(s) by which AR controls clinically relevant gene expression in CaP cells can be manipulated for therapeutic intervention. The objective here is to determine the therapeutic potential of disrupting AR-responsive PKN1-SRF interactions. The central hypothesis is that targeting PKN1 will prevent CaP progression mediated by AR-responsive SRF action. This hypothesis is formulated based on preliminary work produced in the applicant's laboratory. The rationale for the proposed studies is that, once it is understood how AR regulation of SRF action in CaP occurs, select targeting of AR action that is relevant to disease progression will be possible. The central hypothesis is tested by pursuing 3 specific aims: 1) determine the role of PKN1 in the AR-responsive SRF transcriptional complex; 2) define the substrate for PKN1's kinase action in the AR-dependent SRF transcriptional complex; and 3) determine therapeutic potential of targeting AR- responsive PKN1-SRF interactions. Aim 1 will determine the impact of genetic or pharmacological inactivation of PKN1 on the composition and function of AR-dependent SRF complex at target genes using a combination of RNA-Seq, ChIP-Seq and ATAC-Seq assays. In Aim 2, state-of-the-art biotin-based proximity ligation assays and mass spectrometry in CaP models that express wild-type or kinase-dead PKN1 or in which PKN1 action is pharmacologically inhibited will define PKN1-dependent phosphorylation of SRF, the SRF transcriptional complex and its chromatin environment. Aim 3 will determine the therapeutic potential of inhibiting PKN1-SRF interactions in clinically relevant PDXs and fresh ex vivo explants of CaP tissue obtained from patients. Clinically applicable biomarkers of response to such treatment will be sought using gene expression and proximity ligation assays. The proposed research is innovative because it focuses on an entirely different approach to target AR action in CaP: unraveling and targeting an AR-dependent signaling pathway downstream of AR that controls the expression of genes that are relevant for CaP progression. This contribution is significant because is it the first step in a continuum of research that is expected to lead to the development of novel treatment modalities that target specifically AR-mediated gene expression that underlies the lethal CaP phenotype. With respect to expected outcomes, the proposed studies will identify the molecular mechanisms by which PKN1 introduces a therapeutic vulnerability in the AR-PKN1-SRF signaling cascade that gives rise to the aggressive CaP phenotype. These results will have an important positive impact because they will fundamentally advance knowledge about AR action in CaP, in general, and provide new targets for CaP therapy, specifically.